HECT and RING finger families of E3 ubiquitin ligases at a glance

Abstract

The post-translational attachment of ubiquitin, a highly conserved 76-amino-acid polypeptide, directs myriad eukaryotic proteins to a variety of fates and functions. Ubiquitylation is best-known for targeting proteins for degradation by the 26S proteasome. Other functions include internalization and lysosomal targeting, modulation of protein interactions, alteration of subcellular distribution, regulation of transcription, DNA repair and propagation of transmembrane signaling, most notably in the NF-κB pathway. Not surprisingly, ubiquitylation has been linked to virtually every cellular process.Ubiquitylation occurs through the sequential action of three classes of protein: ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s, also referred to as ubiquitin carrier proteins or UBCs), and ubiquitin-protein ligases (E3s) (Komander, 2009). The C-terminal carboxyl group of ubiquitin is activated in an ATP-dependent process, which results in a high-energy thioester linkage with the active-site cysteine of E1. Ubiquitin is trans-thiolated from the E1 to the active-site cysteine of one of ~40 E2s (in mammals). Finally, ubiquitin is generally transferred from the E2 to the ε-amino group of a substrate lysine in an E3-dependent manner. For the E3 ligases of the homologous to the E6AP carboxyl terminus (HECT) domain family (Rotin and Kumar, 2009), this involves an obligate thioester intermediate with the active-site cysteine of the E3. The vast majority of E3 ligases belong to the group of really interesting new gene (RING) and RING-related E3s – such as plant homeodomain (PHD) and leukemia associated protein (LAP) finger proteins, and members of the U-box family (Deshaies and Joazeiro, 2009). These mediate the direct transfer of ubiquitin from E2 to substrate. Ubiquitin can also be added to the N-terminus of proteins (Ciechanover and Ben-Saadon, 2004), and the modification of other nucleophilic amino acids (threonine, serine, cysteine) by ubiquitylation has been described (Cadwell and Coscoy, 2005; Carvalho et al., 2007; Ishikura et al., 2010; Shimizu et al., 2010; Tait et al., 2007; Wang et al., 2007; Williams et al., 2007).Substrates can be modified by a single ubiquitin on one or more sites (monoubiquitylation and multi-monoubiquitylation, respectively) or can be tagged with chains of ubiquitin (poly- or multiubiquitylation). The majority of experimental evidence suggests that chains are built sequentially, beginning with the substrate-attached ubiquitin, although there is evidence for chains being built on E2 or E3 and transferred to substrates en bloc (Bazirgan and Hampton, 2008; Li et al., 2007; Ravid and Hochstrasser, 2007; Wang and Pickart, 2005). Chain formation can occur through the seven internal lysine residues on ubiquitin or its N-terminus (Vijay-Kumar et al., 1987; Behrends and Harper, 2011; Clague and Urbe, 2010). The type of ubiquitin modification specifies the function of the modification (i.e. monoubiquitylation or different chain linkages), together with other factors such as ubiquitin receptors and the opposing action of deubiquitylating enzymes (DUBs); see also the Commentary by Michael J. Clague, Judy M., Coulson and Sylvie Urbé in the previous issue (J. Cell Sci.125, xxxx).Ubiquitylation occurs with exquisite spatial, temporal and substrate specificity, much of which is dictated by the more than 600 E3s that are estimated to be encoded by the mammalian genome (Li et al., 2008). In addition to this cellular specificity and complexity, E3s are implicated in a number of pathophysiological conditions, which makes them attractive therapeutic targets (Kirkin and Dikic, 2011; Lipkowitz and Weissman, 2011). This Cell Science at a Glance article and the accompanying poster describe structural aspects of E3s, and highlight several mammalian pathways and cellular processes in which E3s have key roles. The roles of other specific E3s and cellular processes that are regulated by ubiquitylation are discussed in detail in accompanying review articles in this and the previous issue.In mammals, there are ~30 HECT domain E3s. Among their many functions, HECT E3s have prominent roles in protein trafficking, the immune response, and in several signaling pathways that regulate cellular growth and proliferation (Rotin and Kumar, 2009). The conserved HECT domain (which comprises ~350 amino acids) is located at the C-terminus of these enzymes, whereas their N-terminal domains are diverse and mediate substrate targeting. The HECT domain itself is bi-lobed, consisting of an N-terminal N-lobe that interacts with the E2 and a C-terminal C-lobe that contains the active-site cysteine that forms the thioester with ubiquitin (Huang et al., 1999). The crystal structure of the NEDD4L HECT domain in complex with ubiquitin-conjugated E2 [UBCH5B~Ub, officially known as UBE2D2] shows the C-lobe contacting the esterified ubiquitin and folding down onto UbcH5B, thereby making the distance between the E2 and E3 cata